RESUMEN
Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.
Asunto(s)
Polaridad Celular , Clatrina/metabolismo , Células Epiteliales/citología , Animales , Catepsina D/metabolismo , Línea Celular , Clatrina/deficiencia , Clatrina/genética , Cadenas Pesadas de Clatrina/genética , Cadenas Pesadas de Clatrina/metabolismo , Perros , Células Epiteliales/metabolismo , Aparato de Golgi/metabolismo , Humanos , Inulina/metabolismo , Lisosomas/metabolismo , Transporte de Proteínas , Receptores de LDL/metabolismo , Receptores de Transferrina/metabolismo , Uniones Estrechas/metabolismo , Factores de Tiempo , Red trans-Golgi/metabolismoRESUMEN
Many solute transporters are heterodimers composed of non-glycosylated catalytic and glycosylated accessory subunits. These transporters are specifically polarized to the apical or basolateral membranes of epithelia, but this polarity may vary to fulfill tissue-specific functions. To date, the mechanisms regulating the tissue-specific polarity of heteromeric transporters remain largely unknown. Here, we investigated the sorting signals that determine the polarity of three members of the proton-coupled monocarboxylate transporter (MCT) family, MCT1, MCT3 and MCT4, and their accessory subunit CD147. We show that MCT3 and MCT4 harbor strong redundant basolateral sorting signals (BLSS) in their C-terminal cytoplasmic tails that can direct fusion proteins with the apical marker p75 to the basolateral membrane. In contrast, MCT1 lacks a BLSS and its polarity is dictated by CD147, which contains a weak BLSS that can direct Tac, but not p75 to the basolateral membrane. Knockdown experiments in MDCK cells indicated that basolateral sorting of MCTs was clathrin-dependent but clathrin adaptor AP1B-independent. Our results explain the consistently basolateral localization of MCT3 and MCT4 and the variable localization of MCT1 in different epithelia. They introduce a new paradigm for the sorting of heterodimeric transporters in which a hierarchy of apical and BLSS in the catalytic and/or accessory subunits regulates their tissue-specific polarity.
Asunto(s)
Polaridad Celular/fisiología , Epitelio/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Señales de Clasificación de Proteína/fisiología , Transporte de Proteínas/fisiología , Secuencia de Aminoácidos , Animales , Basigina/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Clatrina/metabolismo , Técnicas de Silenciamiento del Gen , Fusión de Membrana/fisiología , Proteínas de la Fusión de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Transportadores de Ácidos Monocarboxílicos/químicaRESUMEN
Adenoviruses invading the organism via normal digestive or respiratory routes require the Coxsackie-adenovirus receptor (CAR) to infect the epithelial barrier cells. Because CAR is a component of tight junctions and the basolateral membrane and is normally excluded from the apical membrane, most epithelia are resistant to adenoviruses. However, we discovered that a specialized epithelium, the retinal pigment epithelium (RPE), anomalously expressed CAR at the apical surface and was highly susceptible to adenovirus infection. These properties of RPE cells correlated with the absence of the epithelial-specific clathrin adaptor AP1B. Furthermore, knockdown of this basolateral sorting adaptor in adenovirus-resistant MDCK cells promoted apical localization of CAR and increased dramatically Adenovirus infectivity. Targeting assays showed that AP1B is required for accurate basolateral recycling of CAR after internalization. AP1B knock down MDCK cells missorted CAR from recycling endosomes to the apical surface. In summary, we have characterized the cellular machinery responsible for normal sorting of an adenovirus receptor and illustrated how tissue-specific variations in such machinery result in drastic changes in tissue-susceptibility to adenoviruses.
Asunto(s)
Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Adenoviridae/patogenicidad , Clatrina/metabolismo , Células Epiteliales/virología , Subunidades beta de Complejo de Proteína Adaptadora/deficiencia , Animales , Línea Celular , Polaridad Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Perros , Endocitosis , Células Epiteliales/citología , Células Epiteliales/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Transporte de Proteínas , Receptores Virales/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/virología , Uniones EstrechasRESUMEN
CD147, a type I integral membrane protein of the immunoglobulin superfamily, exhibits reversed polarity in retinal pigment epithelium (RPE). CD147 is apical in RPE in contrast to its basolateral localization in extraocular epithelia. This elicited our interest in understanding the basolateral sorting signals of CD147 in prototypic Madin-Darby canine kidney (MDCK) cells. The cytoplasmic domain of CD147 has basolateral sorting information but is devoid of well-characterized basolateral signals, such as tyrosine and di-leucine motifs. Hence, we carried out systematic site-directed mutagenesis to delineate basolateral targeting information in CD147. Our detailed analysis identified a single leucine (252) as the basolateral targeting motif in the cytoplasmic tail of CD147. Four amino acids (243-246) N-terminal to leucine 252 are also critical basolateral determinants of CD147, because deletion of these amino acids leads to mistargeting of CD147 to the apical membranes. We ruled out the involvement of adaptor complex 1B (AP1B) in the basolateral trafficking of CD147, because LLC-PK1 cells lacking AP1B, target CD147 basolaterally. At variance with MDCK cells, the human RPE cell line ARPE-19 does not distinguish between CD147 (WT) and CD147 with leucine 252 mutated to alanine and targets both proteins apically. Thus, our study identifies an atypical basolateral motif of CD147, which comprises a single leucine and is not recognized by RPE cells. This unusual basolateral sorting signal will be useful in unraveling the specialized sorting machinery of RPE cells.
Asunto(s)
Antígenos CD/química , Antígenos CD/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Complejo 1 de Proteína Adaptadora/metabolismo , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Basigina , Línea Celular , Polaridad Celular , Secuencia Conservada , Perros , Células Epiteliales/citología , Células Epiteliales/metabolismo , Semivida , Humanos , Células LLC-PK1 , Leucina/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Epitelio Pigmentado Ocular/citología , Señales de Clasificación de Proteína/genética , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , PorcinosRESUMEN
Electroporation-mediated delivery of molecules is a procedure widely used for transfecting complementary DNA in bacteria, mammalian and plant cells. This technique has proven very efficient for the introduction of macromolecules into cells in suspension culture and even into cells in their native tissue environment, e.g. retina and embryonic tissues. However, in spite of several attempts to date, there are no well-established procedures to electroporate polarized epithelial cells adhering to a tissue culture substrate (glass, plastic or filter). We report here the development of a simple procedure that uses available commercial equipment and works efficiently and reproducibly for a variety of epithelial cell lines in culture.
Asunto(s)
Diferenciación Celular/fisiología , ADN/metabolismo , Electroporación , Células Epiteliales/metabolismo , Proteínas/metabolismo , Transfección/métodos , Animales , Células CACO-2 , Línea Celular , Perros , Células Epiteliales/citología , HumanosRESUMEN
The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (mu1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). Mu1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that mu1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.5 d, sorted TfR correctly in the biosynthetic route but incorrectly in the recycling route. In contrast, these same cells missorted vesicular stomatitis virus G apically in the biosynthetic route. Strikingly, recently confluent MDCK cells (1-3 d) displayed AP1B-dependence in the biosynthetic route of TfR, which decreased with additional days in culture. Sucrose density gradient analysis detected AP1B predominantly in TfR-rich endosomal fractions in MDCK cells confluent for 1 and 4 d. Our results are consistent with the following model: AP1B sorts basolateral proteins in both biosynthetic and recycling routes of MDCK cells, as a result of its predominant functional localization in recycling endosomes, which constitute a post-Golgi station in the biosynthetic route of some plasma membrane proteins. TfR utilizes a direct route from Golgi to basolateral membrane that is established as the epithelial monolayer matures.
Asunto(s)
Subunidades beta de Complejo de Proteína Adaptadora/fisiología , Subunidades beta de Complejo de Proteína Adaptadora/química , Animales , Línea Celular , Membrana Celular/metabolismo , Perros , Endosomas/metabolismo , Células Epiteliales/metabolismo , Aparato de Golgi/metabolismo , Modelos Biológicos , Péptidos/química , Fenotipo , Transporte de Proteínas , ARN Interferente Pequeño/metabolismo , Receptores de Transferrina/metabolismo , Receptores de Transferrina/fisiología , Factores de TiempoRESUMEN
Proton-coupled monocarboxylate transporters (MCT) MCT1, MCT3, and MCT4 form heterodimeric complexes with the cell surface glycoprotein CD147 and exhibit tissue-specific polarized distributions that are essential for maintaining lactate and pH homeostasis. In the parenchymal epithelia of kidney, thyroid, and liver, MCT/CD147 heterocomplexes are localized in the basolateral membrane where they transport lactate out of or into the cell depending on metabolic conditions. A unique distribution of lactate transporters is found in the retinal pigment epithelium (RPE), which regulates lactate levels of the outer retina. In RPE, MCT1/CD147 is polarized to the apical membrane and MCT3/CD147 to the basolateral membrane. The mechanisms responsible for tissue-specific polarized distribution of MCTs are unknown. Here, we demonstrate that CD147 carries sorting information for polarized targeting of the MCT1/CD147 hetero-complexes in kidney and RPE cells. In contrast, MCT3 and MCT4 harbor dominant sorting information that cotargets CD147 to the basolateral membrane in both epithelia. RNA interference experiments show that MCT1 promotes CD147 maturation. Our results open a unique paradigm to study the molecular basis of tissue-specific polarity.